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Biological systems are constantly exposed to electromagnetic fields (EMFs) in the form of natural geomagnetic fields and EMFs emitted from technology. While strong magnetic fields are known to change chemical reaction rates and free radical concentrations, the debate remains about whether static weak magnetic fields (WMFs; <1 mT) also produce biological effects. Using the planarian regeneration model, we show that WMFs altered stem cell proliferation and subsequent differentiation via changes in reactive oxygen species (ROS) accumulation and downstream heat shock protein 70 (Hsp70) expression. These data reveal that on the basis of field strength, WMF exposure can increase or decrease new tissue formation in vivo, suggesting WMFs as a potential therapeutic tool to manipulate mitotic activity.
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Van Huizen et al., Sci. Adv. 2019; 5 : eaau7201 30 January 2019
SCIENCE ADVANCES | RESEARCH ARTICLE
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BIOPHYSICS
Weak magnetic fields alter stem cell–mediated growth
Alanna V. Van Huizen1, Jacob M. Morton1, Luke J. Kinsey1,
Donald G. Von Kannon1, Marwa A. Saad1, Taylor R. Birkholz1, Jordan M. Czajka1,
Julian Cyrus2, Frank S. Barnes2, Wendy S. Beane1*
Biological systems are constantly exposed to electromagnetic fields (EMFs) in the form of natural geomagnetic
fields and EMFs emitted from technology. While strong magnetic fields are known to change chemical reaction
rates and free radical concentrations, the debate remains about whether static weak magnetic fields (WMFs; <1 mT)
also produce biological effects. Using the planarian regeneration model, we show that WMFs altered stem cell
proliferation and subsequent differentiation via changes in reactive oxygen species (ROS) accumulation and
downstream heat shock protein 70 (Hsp70) expression. These data reveal that on the basis of field strength, WMF
exposure can increase or decrease new tissue formation in vivo, suggesting WMFs as a potential therapeutic tool
to manipulate mitotic activity.
INTRODUCTION
Exposure to electromagnetic fields (EMFs) occurs both from modern
technology and Earth’s natural geomagnetic field, which averages
25 to 65 T (1). In many circles, it is assumed that the quantum of
energy associated with these weak magnetic fields (WMFs; <1 mT)
is too insubstantial to be biologically important (2). Despite the fact
that stronger magnetic fields are known to affect chemical reaction
rates and free radical concentrations (3,4), initial studies of WMF
effects on cell cultures produced contradictory results. While one
study reported increased levels of the transcription factor c-Myc in
human leukemia cells following WMF exposure, a different group
failed to replicate these results (5,6). Another study showed that
WMFs stimulated protein tyrosine kinases Lyn and Syk levels in
B-lineage lymphoid cells, while two later studies found no signifi-
cant differences (7–9). However, recent evidence indicates that WMFs
can affect biological systems in multiple ways. WMF exposure in-
creased intracellular calcium concentrations and the rate of cellular
development in satellite cells, and caused embryo mortality as well
as altered vertebrae development in roach embryos (10,11). Cell-
dependent effects from WMFs were seen in rat renal versus cortical
astrocyte cells, with decreased levels of apoptosis, proliferation, and
necrosis in renal cells but increases in all three in astrocyte cells (12).
WMFs were also found to produce transient induction of the mem-
brane permeability transition and increased cytosolic cytochrome c
levels in human amniotic cells via an increase in reactive oxygen
species (ROS) (13).
A theoretical basis exists for the effects of WMFs on the concen-
tration of free radicals such as ROS, as outlined in (14–16). Tradi-
tionally viewed as harmful, ROS can trigger cell death and thus are
highly regulated by antioxidant enzymes such as superoxide dis-
mutase (SOD), but ROS are also beneficial—acting as regulatory
mediators (17), assisting in muscle repair (18), and modulating cell
signaling (19). More recently, ROS signaling has been shown to reg-
ulate new tissue growth, as such in zebrafish where ROS production
triggers apoptosis-induced compensatory proliferation required for
regeneration (20).
In this study, we sought to determine whether WMFs could pro-
duce biological effects in vivo (in whole organisms) using the robust
planarian regeneration model. Planaria are free-living flatworms
that are capable of regenerating all tissues, including the central
nervous system and brain, owing to a large adult stem cell (ASC)
population that comprises ~25% of all cells (21). After injury, ASCs
mount an animal-wide proliferative response that initially peaks at
~4hours; this is followed by ASC migration to the wound site over
the first 72hours, when a second mitotic peak occurs (22). This
activity produces the blastema, a collection of unpigmented ASC
progeny that forms the core of new tissues. Full regeneration of
missing structures occurs in 2 to 3weeks through the combination
of new tissue growth and the apoptotic remodeling and scaling of
old tissues.
RESULTS AND DISCUSSION
To determine whether WMFs affect tissue growth during planarian
regeneration, we amputated animals above and below the pharynx
(feeding tube) and examined blastema outgrowth at 3days postam-
putation (dpa) (Fig.1A) following WMF exposure. The setup of our
magnetic field apparatus is outlined in fig. S1. We found that 200 T
WMF exposure produced blastema sizes that were significantly re-
duced as compared to both untreated and Earth-normal 45 T
field strength controls (Fig.1, C and D). Temporal analyses, where
regenerates were exposed for different lengths of time during the
first 72hours of regeneration (Fig.1B), revealed that 200 T ex-
posure was required early and must be maintained throughout
blastema formation to affect growth [24hours postamputation
(hpa) to 3 dpa]. Because shorter, single-day exposures failed to
affect blastema size, these data suggest the presence of recovery
mechanisms to ensure initiation of new growth. Furthermore, we
found that WMFs produced field strength–dependent effects:
Significant reductions of blastema size were observed from 100 to
400 T, but conversely, a significant increase in outgrowth occurred
at 500 T (Fig.1E).
We hypothesized that WMF effects were due to altered ROS
levels, which peak at the wound site by 1 hpa and are required for
planarian blastema formation (23). Pharmacological ROS inhibition
resulted in significantly reduced blastema sizes (Fig.2, A and B),
phenocopying 200 T WMF exposure. To determine whether WMF
1Department of Biological Sciences, Western Michigan University, Kalamazoo, MI
49008, USA. 2Department of Electrical, Computer, and Energy Engineering, Uni-
versity of Colorado Boulder, Boulder, CO 80309, USA.
*Corresponding author. Email: wendy.beane@wmich.edu
Copyright © 2019
The Authors, some
rights reserved;
exclusive licensee
American Association
for the Advancement
of Science. No claim to
original U.S. Government
Works. Distributed
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Commons Attribution
License 4.0 (CC BY).
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exposure altered ROS levels, we used a cell-permeant fluorescent
general oxidative stress indicator dye to examine ROS accumula-
tion during regeneration. Our results revealed that ROS levels were
significantly reduced and/or absent from the wound site after both
200 T exposure and direct ROS inhibition, as compared to con-
trols at 1 hpa (Fig.2C). Furthermore, increasing ROS levels via SOD
inhibition by RNA interference (RNAi) was sufficient to completely
rescue regenerative outgrowth in 200 T–exposed regenerates
(Fig.1D and fig. S2, A and B). These data suggest that WMF effects
on new tissue production are largely due to manipulation of ROS
levels in vivo. We also found that SOD inhibition alone was suffi-
cient to significantly increase blastema sizes in 45 T controls
(Fig.2D), suggesting that tissue growth is highly dose dependent on
ROS levels. This is supported by measurements at 500 T, which
also resulted in increased growth, that revealed increased ROS levels
(average signal intensity of 61.7 for 500 T versus 17.7 for 45 T
controls; n=12; P<0.01 by Student’s t test).
To investigate genetic mechanisms by which ROS levels (and
thus WMFs) regulate regenerative outgrowth, we examined their
effects on heat shock protein 70 (Hsp70) expression. Hsp70 is a
stress response protein that acts as a chaperone for protein folding
during repair, promoting both normal cell survival and cancer cell
growth (24). ROS have been shown to affect Hsp70 expression in cell
culture (25), and cadmium exposure (which decreases SOD activity
and thus increases ROS levels) alters expression of heat shock pro-
teins in a dose-dependent manner (26). Our results demonstrate
that Hsp70 inhibition by RNAi significantly reduced blastema sizes
during planarian regeneration (Fig.3, A and B), similar to 200 T
WMF exposure and direct ROS inhibition. Furthermore, Hsp70
expression was lost following both 200 T exposure and direct ROS
inhibition (Fig.3, C and D). Consistent with these data, increasing
Fig. 1. WMFs alter planarian regeneration. (A) Composite image illustrating
Schmidtea mediterranea amputation scheme. (B) Temporal analyses of 200 T WMF
exposure on anterior blastema size. Each row represents an experimental group of
pharynx fragments that were exposed at the indicated times and scored at 3 dpa.
The length of each bar is the duration of 200 T exposure. Red bars, blastema in-
hibition (Student’s t test against 45 T; P ≤ 0.05). Gray bars, no effect. n ≥ 12 for all
conditions. (C and D) Blastema size following 200 T exposure versus untreated
and 45 T controls. Arrowheads indicate presence (solid) or lack (open) of blastema.
Scale bars, 200 m. One-way analysis of variance (ANOVA) with Tukey’s multiple
comparison test; n ≥ 24. (E) Blastema size following exposure to different field
strengths. Student’s t test against 45 T; n ≥ 16. Red bars, reduced blastema size.
Green bar, increased blastema size. Gray bars, no effect. For all: **P < 0.01, ***P < 0.001,
and ****P < 0.0001; error bars are SEM; anterior is up; and animals scored at 3 dpa.
Fig. 2. WMFs affect ROS levels during early regeneration. (A and B) Pharmaco-
logical ROS inhibition using 10 M diphenyleneiodonium chloride (DPI) scored at
3 dpa. Student’s t test; n 20. Scale bars, 200 m. DMSO, dimethyl sulfoxide. (C) An-
terior ROS accumulation detection 1 hpa using the general oxidative stress indicator
dye 5-(and-6)-chloromethyl-2,7-dicholorodihydrofluorescein diacetate (CM-H2D-
CFDA). One-way ANOVA with Tukey’s multiple comparison test; n ≥ 15. Scale bars,
200 m. (D) RNAi of SOD imaged 3 dpa. Student’s t test against 45 T; n ≥ 10. Scale
bars, 100 m. Red bar, reduced blastema size. Green bar, increased blastema size.
Gray bar, no effect. For all: Solid arrowheads indicate normal blastemas; open
arrowheads, lack of blastema; and double arrowheads, increased blastema; **P < 0.01
and ****P < 0.0001; error bars are SEM; and anterior is up.
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ROS levels via SOD RNAi was sufficient to rescue Hsp70 expres-
sion in 200 T–exposed regenerates (fig. S2C). These data sug-
gest that increased ROS levels lead to increased Hsp70 expression
during planarian regeneration and that WMFs can alter both pro-
cesses in vivo.
To determine whether the observed changes in blastema size
were due to changes in proliferation, we examined mitotic activity
via phospho–histone H3 (pH3) staining at the wound site at 4 hpa.
Our data revealed that 200 T WMF exposure, direct ROS inhibi-
tion, and direct Hsp70 inhibition all resulted in significantly re-
duced mitotic activity as compared to control conditions (Fig.3E).
In planarians, ASCs are the only mitotically active cells, suggesting
that WMFs (through ROS and Hsp70) affect stem cell activity. We
used a planarian ASC marker (Piwi) to examine stem cell population
levels during regeneration, as well as a late-progeny marker (AGAT)
to examine stem cell differentiation. We found that 200 T WMF
exposure, direct ROS inhibition, and direct Hsp70 inhibition all re-
sulted in significantly reduced ASC levels and stem cell differentia-
tion near the blastema at 3 dpa (Fig.4, A and B). Together, these
data suggest that WMFs are able to alter stem cell regulation during
regeneration via changes in ROS signaling.
Our data confirm that WMFs affect biological systems and es-
tablish a nascent mechanistic pathway by which this occurs. Cur-
rently, the main hypothesis for how magnetic fields interact with
biological systems is through radical pair recombination (Fig.4C)
(1,3,27). In this model, components of a parent molecule can disso-
ciate into a radical pair. Each unpaired electron will have opposing
valence spin directions but may undergo a shift in spin direction.
Antiparallel valence electron spins (singlet state) allow quick recom-
bination of radicals back into the parent molecule. Alternatively,
parallel spin states (triplet state) prevent recombination, providing
sufficient time for the pair to diffuse away from one another, creat-
ing free radicals (3). Our data suggest that WMF exposure promotes
singlet or triplet states depending on field strength, which results in
decreased or increased ROS concentrations, respectively.
Our data reveal an underlying pathway by which WMFs affect
planarian regeneration (Fig.4D). WMF exposure alters ROS levels,
Fig. 3. WMF effects on new tissue growth are caused by changes in both Hsp70
expression and proliferation. (A and B) Hsp70 RNAi scored at 3 dpa. Student’s
t test; n ≥ 15. Arrowheads indicate presence (solid) or lack (open) of blastema. Con-
trol RNA: Venus-GFP. Scale bars, 200 m. (C) Untreated intact animal whole-mount
in situ hybridization (WISH) with the Hsp70 probe (n = 13). Scale bar, 200 m. (D) Ef-
fects on Hsp70 expression visualized by WISH at 3 dpa. The anterior region is shown
(n5). Scale bars, 100 m. (E) Phospho–histone H3 (pH3) staining of whole regen-
erates at 4 hpa. Only the anterior region is shown in the images. One-way ANOVA with
Tukey’s multiple comparison test; n ≥ 6. Scale bars, 50 m. For all: DPI used at 10 M;
**P < 0.01, ***P < 0.001, and ****P < 0.0001; error bars are SEM; and anterior is up.
Fig. 4. WMFs affect stem cell regulation during early regeneration. (A and B) Fluo-
rescence in situ hybridization at 3 dpa to examine (A) the stem cell population (Piwi probe ;
n6) and (B) stem cell differentiation (AGAT probe; n 5). The anterior region is
shown. DPI used at 10 M. Top panels are significantly different from bottom panels
(Student’s t test; P0.01). Scale bars, 50 m. (C) Model for WMF effects on radical pair
recombination. (D) Proposed pathway for 200 T WMF effects on planarian regeneration.
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which lead to changes in Hsp70 expression, which has conse-
quences on stem cell proliferation and subsequent differentia-
tion regulating blastema formation. It is likely that the effects on
differentiation are the result of reduced numbers of proliferating
stem cells, although direct effects cannot be ruled out. These find-
ings are consistent with recent research highlighting the impor-
tance of ROS signaling in the cell in general (13,1719) and in
regeneration specifically (20,23,28,29). In addition, these data
are consistent with studies that have linked EMF exposure to both
increased Hsp70 expression and increased regeneration (3032).
Previous studies have also shown the importance of ROS signal-
ing in initiating apoptotic-induced compensatory proliferation
during regeneration (20). While our data demonstrate a link be-
tween WMFs and ROS-mediated stem cell proliferation, it is possi-
ble that effects on stem cell migration to the wound site and/or
on apoptosis are also involved. Thus, future studies should investi-
gate these mechanisms as possibilities for WMF effects on stem cell
activity.
The ability of WMFs to modulate regenerative outgrowth in vivo
suggests that WMFs could be a potential therapeutic tool. In sup-
port of this, our investigations with mouse fibroblast cells revealed
that WMF exposure caused reduced growth of fibrosarcoma cell
cultures but had no effect on noncancerous fibroblast controls (33).
Together, these data suggest that highly proliferative cell popula-
tions may be specifically targeted during WMF exposure. If true,
this would suggest novel possibilities for cancer treatments, where
improved methods are needed to inhibit tumor growth while
leaving surrounding cells unaffected.
MATERIALS AND METHODS
Animal care and amputations
An asexual clonal line of Schmidtea mediterranea (CIW4) was main-
tained at 18°C in the dark. All planarians were kept in worm water;
worm water consists of Instant Ocean salts (0.5g/liter) in ultrapure
water of Type 1. Animals were fed no more than once a week with
“natural” (no antibiotics or hormones) liver paste made from whole
calf liver (Creekstone Farms). Liver was frozen and thawed only once
before feeding animals. Worms 5 to 7mm in length were starved at
least 1 week before experimentation. S. mediterranea were amputated
into trunk fragments via scalpel cuts made just above and below the
pharynx. Amputations were done under a dissecting microscope
on a custom-made cooling Peltier plate, as previously described
(34). Untreated control animals were allowed to regenerate in stan-
dard biological oxygen demand incubators (VWR) at 18°C without
light, which is the normal method for the planarian field.
Magnetic field apparatus
A magnetic field enclosure (MagShield box) was constructed of
-metal (which blocks magnetic fields) with a vertical -metal par-
tition for running parallel experiments (control and experimental).
Two custom-built triaxial Helmholtz coils were positioned in the
exact center of each partition (via a stack of plastic well plates) to
ensure that experiments were uniformly exposed to each specific
magnetic field. Each coil was composed of a Plexiglas skeleton
around which a ceramic-insulated copper wire was wrapped multi-
ple times running in two parallel strands on each of the x, y, and
z axes. Direct electric current to each coil was supplied by DC power
sources (Mastech HY3005D-3) and was fed through the x and
y coils in order to create a uniform static WMF. Before each exper-
iment, Helmholtz coils were characterized using a Gauss meter
(AlphaLab models GM1-ST or GM1-HS), which was also used to
verify the magnetic field at the end of each experiment.
Magnetic field exposure assay
The MagShield box was housed in a temperature-controlled room
(20°C). The temperature inside the Helmholtz coils during mag-
netic field exposure assays was randomly tested twice a day over the
3-day assay, with an average temperature of 22°C (±1°C). Animals
were placed in worm water in either 35 or 60 mm petri dishes at the
indicated times into the MagShield box in the center of the partition
(via a stack of larger plastic petri dishes). Animals were exposed to
controlled static magnetic fields (in the dark) always in tandem:
with one side of the MagShield partition set to Earth-normal 45 T
(for controls) and the other partition set to the indicated experi-
mental WMF strength. Unless otherwise indicated, experimental
planarians were exposed to 200 T from 5minutes postamputation
(mpa) to 72hpa. Experiments were repeated three times (except for
untreated controls, which were performed once). Total biological
replicates for each condition were as follows: untreated, n=24;
45 T, n=29; and 200 T, n=25. For the temporal trials, planarians
were exposed beginning at 0 hpa (i.e., 5 mpa), 30 mpa, 1 hpa,
2.5 hpa, 24 hpa, and 48 hpa until the end of the 72-hour period.
In addition, planarians were exposed beginning at 0 hpa and ending
at 12, 24, or 48 hpa, at which time they were removed from the
MagShield box and allowed to continue regenerating in standard
incubators (as for untreated controls) until the end of the 72-hour
period. Last, planarians were preexposed starting 24hours before
amputation and were either allowed to regenerate with no further
exposure or returned to the MagShield box to be further exposed
for either 24 or 48 hpa (after which time they continued regen-
erating in standard incubators until the end of the 72-hour period).
Temporal experiments were performed once (except for the 0- to
72-hour exposure, which was repeated three times, and the 30-min
to 72-hour exposure, which was repeated twice). Total biological
replicates for each condition were as follows: 0 to 72hours, n=25;
30min to 72hours, n=19; 1 to 72hours, n= 12; 2.5 to 72hours,
n=14; 24 to 72hours, n=17; 48 to 72hours, n=16; 0 to 12hours,
n=18; 0 to 24hours, n=20; 0 to 48hours, n=17; −24 to
24 hours, n=13; −24 to 48hours, n=20; and−24 to 0hours (ampu-
tation), n=15. For the field strength trials, experiments were per-
formed once, except for 200 T, which was repeated three times,
and 45 T, which was repeated eight times (as every field strength
was run with concurrent controls). Total biological replicates for
each condition were as follows: 45 T, n=119; 0 T, n=30; 100 T,
n=28; 200 T, n=25; 300 T, n=18; 400 T, n=18; 500 T, n=17;
and 600 T, n=16.
Pharmacological inhibition of ROS
ROS accumulation was inhibited with diphenyleneiodonium chlo-
ride (DPI; Sigma D2926). Animals were presoaked in 10M DPI
[made from a 3mM dimethyl sulfoxide (DMSO) stock] for 5 hours
before amputation. Newly amputated pharynx fragments were im-
mediately returned to 10M DPI and were allowed to regenerate
until 72 hpa, at which time planarians were imaged for blastema size
analyses. Control experiments were placed in DMSO (vehicle con-
trol). Total biological replicates for each condition were as follows:
DMSO, n=20 and DPI, n=22.
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ROS indicator dye assay
The cell-permeant fluorescent general oxidative stress indicator dye,
5-(and-6)-chloromethyl-2,7-dicholorodihydrofluorescein diacetate
(CM-H2DCFDA; Molecular Probes C6827), was used to visualize
ROS accumulation (excitation, 470nm; emission, 525nm). One hour
before imaging, worms were incubated in 25M CM-H2DCFDA
made from 10mM DMSO stock. For WMF experiments, following
a 23-hour WMF preexposure period, whole worms were removed
from the MagShield box, cut into pharynx fragments, placed into
25M CM-H2DCFDA, and returned to the MagShield box for the
1-hour incubation period. For ROS inhibition experiments, follow-
ing a 5-hour pretreatment period, whole worms were removed from
DPI, cut into pharynx fragments, and then placed into a combination
of 10M DPI plus 25M CM-H2DCFDA for the 1-hour incubation
period. After the 1-hour CM-H2DCFDA incubation period, all
worms were rinsed three times in fresh worm water and then the
ventral side was imaged using 35 mm FluoroDishes (WPI FD35-
100) and 25 mm round no. 1.5 coverslips (WPI 503508). Signal in-
tensity at the wound site was normalized to signal intensity of
the central body to control for differences in dye loading between
animals. Experiments were performed twice (except for DMSO
and DPI). Total biological replicates for each condition were as
follows: untreated, n =37; 45 T, n=26; 200 T, n= 24; DMSO,
n=19; and DPI, n=15.
RNA interference
RNAi was performed via feeding of in vitro–synthesized double-
stranded RNAi, as previously described (35). A 489 base pair (bp)
region of S. mediterranea SOD (SMU15011417) was used to generate
SOD RNAi. The primers were 5-ACTGGAGCCATCAATATCTGG
and 3-TAATCCGGCCTTACATTTTTG. A 552 bp region of
S. mediterranea Hsp70 (SMU15039086) was used to generate Hsp70
RNAi. The region was from 5-GGTTTTTGATTTGGGTGGTG to
3-AGCTGTTGCTATGGGAGC. Worms were fed with RNAi three
times over 8 days before being amputated on day 9, as indicated
above. Control RNAi was double-stranded RNA to Venus-GFP,
which is not present in the planarian genome. SOD RNAi rescue
experiments were performed twice (except for 45 T+SOD RNAi).
Total biological replicates for each condition were as follows: 45 T,
n=20; 200 T, n=20; 45 T+SOD RNAi, n=10; and 200 T+SOD
RNAi, n=20. Total biological replicates for each condition for Hsp70
RNAi morphology experiments were as follows: control RNAi, n=15
and Hsp70 RNAi, n=15.
Immunostaining and in situ hybridization
Immunostaining was performed as previously described (34). The
primary antibody used was anti-pH3 (Sigma/Millipore 04-817; 1:25).
The secondary antibody used was goat anti-rabbit horseradish per-
oxidase (Invitrogen 65-6120) with TSA Cyanine 3 (Cy3)–tyramide
amplification (PerkinElmer; 1:50). Total biological replicates for each
condition were as follows: untreated, n=14; control RNAi, n=6;
Hsp70 RNAi, n=14; 45 T, n=14; 200 T, n=13; DMSO, n=12;
and DPI, n=15.
Colorimetric whole-mount in situ hybridization (WISH) was
performed as previously described (35). A 601 bp region of
S. mediterranea SOD was used to generate riboprobe. The region
was from 5-ACAACGGCAATGAACTTATTAATA to 3-TAATCT-
TAATATTGCTCTTGAAC. Total biological replicates for each
condition for the SOD probe were as follows: control RNAi, n=13
and SOD RNAi, n=10. Riboprobe for Hsp70 was generated from
the same region as the RNAi. Total biological replicates for each
condition for the Hsp70 probe were as follows: intact, n= 13; un-
treated, n =13; Hsp70 RNAi, n =12; 45 T, n=5; 200 T, n=5;
DMSO, n=5; DPI, n=7; 200 T+control RNAi, n=10; and
200 T+SOD RNAi, n=8. Fluorescence in situ hybridization was car-
ried out as previously described (36), with the following exceptions:
Both prehybe and hybe used a yeast RNA concentration of 1mg/ml,
and probes were diluted to 0.5ng/l and hybridized for 24hours. A
404 bp region of S. mediterranea AGAT-1 (NB.8.8b) was used to
generate AGAT riboprobe. The primers were 5-GGAGTTAAAGT-
GTCCATCCAG and 3-GTTGCTAACCTGACTGACATGC. A 2461 bp
region of S. mediterranea Piwi-1 (Q2Q5Y9.1) was used to generate
the Piwi riboprobe. The region was from 5-GATCCCAATTTA-
AGACCAAGAAGAG to 3-TTTTTATGTATTCGATTAAAAAAAA.
Total biological replicates for each condition for the Piwi probe
were as follows: untreated, n=7; Hsp70 RNAi, n=9; 45 T, n=7;
200 T, n=7; DMSO, n=6; and DPI, n=6. Total biological repli-
cates for each condition for the AGAT probe were as follows: un-
treated, n =5; Hsp70 RNAi, n=9; 45 T, n=6; 200 T, n=7;
DMSO, n=6; and DPI, n=6.
Image collection
Images were taken using a ZEISS V20 fluorescence stereomicro-
scope with AxioCam MRc or MRm camera and ZEN lite software
(ZEISS). Regenerates were imaged while fully extended and moving
to ensure the absence of any tissue bunching, which could affect
analyses. Heat maps for visualizing intensity of ROS levels were
generated using the standard rainbow lookup table (LUT) within
the ZEN lite software. Adobe Photoshop was used to orient, scale,
and improve clarity of images (but not for fluorescent images). Data
were neither added nor subtracted; original images are available
upon request.
Quantification and statistical analyses
For blastema size, the magnetic lasso tool in Adobe Photoshop was
used to generate total pixel counts for both the anterior and poste-
rior blastemas (as well as the entire regenerate). To control for
worms of different sizes, blastema sizes were expressed as a ratio of
blastema size/total regenerate size. For ROS indicator dye assay, the
magnetic lasso tool was used to obtain mean gray intensity values
for both the anterior and posterior blastema, as well as baseline
mean pixel intensity values (from the center of the regenerate). Signal
intensity was expressed as average blastema pixel intensity − average
baseline mean pixel intensity. For the pH3 assay, images were masked
to avoid background signal and pH3+ cells were counted with the
RTCN tool in ImageJ; the whole regenerate was measured with the
magnetic lasso tool in Adobe Photoshop, and the final counts were
shown as cells per square millimeter. Significance was determined
using either a one-way analysis of variance (ANOVA) with Tukey’s
multiple comparison test (using GraphPad Prism version 7.00 for
Mac) or a two-tailed Student’s t test with unequal variance (using
Microsoft Excel).
SUPPLEMENTARY MATERIALS
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/
content/full/5/1/eaau7201/DC1
Fig. S1. Magnetic field enclosure (MagShield) setup.
Fig. S2. Loss of SOD rescues 200 T WMF exposure by increasing levels of ROS.
on January 31, 2019http://advances.sciencemag.org/Downloaded from
Van Huizen et al., Sci. Adv. 2019; 5 : eaau7201 30 January 2019
SCIENCE ADVANCES | RESEARCH ARTICLE
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Acknowledgments: We thank the Beane laboratory members for comments. Funding: This
research was supported by NSF EAGER grants 1644371 and 1644384 to F.S.B. and W.S.B.,
respectively. Author contributions: J.C. and F.S.B. constructed the MagShield apparatus;
W.S.B. and F.S.B. designed the experiments; J.M.M., L.J.K., J.M.C., D.G.V.K., T.R.B., and W.S.B.
performed the experiments with assistance from M.A.S. and A.V.V.H.; J.M.M., L.J.K., M.A.S.,
A.V.V.H., and W.S.B. analyzed the data; A.V.V.H., W.S.B., and F.S.B. wrote and edited the
manuscript. All authors read and approved the manuscript. Competing interests: The authors
declare that they have no competing interests. Data and materials availability: All data
needed to evaluate the conclusions in the paper are present in the paper and/or the
Supplementary Materials. Additional data related to this paper may be requested from the
authors.
Submitted 9 July 2018
Accepted 17 December 2018
Published 30 January 2019
10.1126/sciadv.aau7201
Citation: A. V. Van Huizen, J. M. Morton, L. J. Kinsey, D. G. Von Kannon, M. A. Saad, T. R. Birkholz,
J. M. Czajka, J. Cyrus, F. S. Barnes, W. S. Beane, Weak magnetic fields alter stem cell–mediated
growth. Sci. Adv. 5, eaau7201 (2019).
on January 31, 2019http://advances.sciencemag.org/Downloaded from
mediated growthWeak magnetic fields alter stem cell
M. Czajka, Julian Cyrus, Frank S. Barnes and Wendy S. Beane
Alanna V. Van Huizen, Jacob M. Morton, Luke J. Kinsey, Donald G. Von Kannon, Marwa A. Saad, Taylor R. Birkholz, Jordan
DOI: 10.1126/sciadv.aau7201
(1), eaau7201.5Sci Adv
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REFERENCES http://advances.sciencemag.org/content/5/1/eaau7201#BIBL
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... Circadian rhythms, for instance, are sensitive to magnetic fields 4,5 . Stem cell development, both neurogenesis and other, is modulated by weak magnetic fields; a phenomenon that is thought to be mediated by reactive oxygen species (ROS) [6][7][8][9] . Magnetic fields have also been shown to be important to a number of different biological functions with further implications. ...
... But experiments show that the human brain also responds to perturbations of the geomagnetic field. Magnetic field changes result in a decrease in amplitude of alpha frequency (8)(9)(10)(11)(12)(13) Hz) brain waves, which are related to the brain's processing of external stimuli 14 . Research also suggests that disruptions to the Earth's field caused by geomagnetic storms correlate with physiological and psychological changes, including increased instances of suicide (though it is unclear whether the effect is due to direct magnetic effects or increased solar radiation) [15][16][17] . ...
... Another investigated whether static weak magnetic fields might change stem cell proliferation and differentiation, through modulation of ROS and heat shock proteins. The results demonstrated different field strengths increased or decreased tissue formation in vivo 9 and furthermore that this specifically involved modulation of superoxide species 66 . Superoxide has also been shown to play a pivotal role in mammalian magnetic field signal transduction relating to circadian rhythms 67 . ...
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For the first time in history, humankind might conceivably begin to imagine itself as a multi-planetary species. This goal will entail technical innovation in a number of contexts, including that of healthcare. All life on Earth shares an evolution that is coupled to specific environmental conditions, including gravitational and magnetic fields. While the human body may be able to adjust to short term disruption of these fields during space flights, any long term settlement would have to take into consideration the effects that different fields will have on biological systems, within the space of one lifetime, but also across generations. Magnetic fields, for example, influence the growth of stem cells in regenerative processes. Circadian rhythms are profoundly influenced by magnetic fields, a fact that will likely have an effect on mental as well as physical health. Even the brain responds to small perturbations of this field. One possible mechanism for the effects of weak magnetic fields on biological systems has been suggested to be the radical pair mechanism. The radical pair mechanism originated in the context of spin chemistry to describe how magnetic fields influence the yields of chemical reactions. This mechanism was subsequently incorporated into the field of quantum biology. Quantum biology, most generally, is the study of whether non-trivial quantum effects play any meaningful role in biological systems. The radical pair mechanism has been used most consistently in this context to describe the avian compass. Recently, however, a number of studies have investigated other biological contexts in which the radical pair might play a role, from the action of anaesthetics and antidepressants, to microtubule development and the proper function of the circadian clock... (full abstract in the manuscript)
... are used for pain management during rehabilitation and with musculoskeletal diseases such as neuropathy and fibromyalgia [10][11][12][13][14]. Research has demonstrated that exposure to even weak MFs can affect biological systems by altering free radical formation-believed to result from MF interactions with spin dynamics [15][16][17][18][19][20][21]. Weak MFs have been shown to change reactive oxygen species (ROS) signaling and alter cellular outcomes in vivo [19,22], and thus they represent a potential means to control host defense/immunological responses, regenerative stem cell proliferation, and cancer progression [23][24][25][26][27][28]. Despite increased interest in uncovering the underlying mechanisms, standardized methods for experimental exposure are lacking and few model systems have been used for in vivo studies (for a recent review, see [29]). ...
... In vitro, we showed that exposure to static MFs of 0.5 μT and 600 μT MFs inhibited HT-1080 fibrosarcoma cell growth in culture, while 300 and 400 μT increased growth [16,17]. In vivo, we demonstrated that static weak MF exposure of regenerating planarians was able to manipulate stem cell proliferation and gene expression (where 200 μT decreased and 200 μT increased new tissue growth) via changes in superoxide accumulation after injury [19,22]. Our current efforts aim to investigate hypomagnetic field (nT) effects in the vertebrate model organism, Xenopus laevis. ...
... This protocol or parts of it has been used and validated in the following research articles: A. Planarian regeneration experiments (Figure 12) 1. Amputation scheme ( Figure 12A): Previously, we investigated static MF effects on planarian regeneration only for trunk fragments (with heads and tails removed) that have two amputation planes (one above and one below the pharynx/feeding tube) [19,22]. To determine the effects on fragments with a single amputation plane, we investigated three other fragment types. ...
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... The Guy Foundation originally became interested in space research because of the potential of quantum biology to explain how life interacts with magnetic fields, for instance, in bird navigation, 49,50 which soon led to researchers finding that altering magnetic fields could manipulate mitochondrial function. [51][52][53] When combined with the discovery that astronauts develop mitochondrial dysfunction 5 , this raised the possibility that QB could be an approach to understand space health. The Foundation is not the only group to suggest that hypomagnetic fields could directly affect metabolism in space. ...
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It has been shown that magnetic fields in the extremely low frequency range (ELF-MF) can act as a stressor in various in vivo or in vitro systems, at flux density levels below those inducing excitation of nerve and muscle cells, which are setting the limits used by most generally accepted exposure guidelines, such as the ones published by the International Commission on Non-Ionizing Radiation Protection. In response to a variety of physiological and environmental factors, including heat, cells activate an ancient signaling pathway leading to the transient expression of heat shock proteins (HSPs), which exhibit sophisticated protection mechanisms. A number of studies suggest that also ELF-MF exposure can activate the cellular stress response and cause increased HSPs expression, both on the mRNA and the protein levels. In this review, we provide some of the presently available data on cellular responses, especially regarding HSP expression, due to single and combined exposure to ELF-MF and heat, with the aim to compare the induced effects and to detect possible common modes of action. Some evidence suggest that MF and heat can act as costressors inducing a kind of thermotolerance in cell cultures and in organisms. The MF exposure might produce a potentiated or synergistic biological response such as an increase in HSPs expression, in combination with a well-defined stress, and in turn exert beneficial effects during certain circumstances.
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